Residues 17 and 18 in nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRPSGDLYERLLRARGETYGRLLGEVEDGYSQSP from residues 13 to 43. The YXXL motifs in this context match very well with consensus sequences for SH2 binding domains and are similar to ones present in nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting PBMC cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pig-tailed monkeys characterized by severe diarrhea, rash and extensive lymphoid proliferation in the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with src in co-transfected COS cells and both 60 kDa src and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype and they provide evidence of a role for nef in signal transduction and cellular activation.